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In search of secreted protein biomarkers for the anti-inflammatory effect of β2-adrenergic receptor agonists: Application of DIGE technology in combination with multivariate and univariate data analysis tools

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Author: Verhoeckx, K.C.M. · Gaspari, M. · Bijlsma, S. · Greef, J. van der · Witkamp, R.F. · Doornbos, R.P. · Rodenburg, R.J.T.
Type:article
Date:2005
Institution: TNO Kwaliteit van Leven
Source:Journal of Proteome Research, 6, 4, 2015-2023
Identifier: 238774
doi: doi:10.1021/pr050183u
Keywords: Pharmacology · Analytical research · β2-adrenergic receptor · Difference gel electrophoresis · Macrophage inflammatory protein-1β · Multivariate data analysis · Partial least squares discriminant analysis · Principal component analysis · Zilpaterol · adenylate kinase · beta 2 adrenergic receptor · beta 2 adrenergic receptor blocking agent · beta 2 adrenergic receptor stimulating agent · biological marker · butyryl cyclic AMP · clenbuterol · endotoxin · Escherichia coli lipopolysaccharide · formoterol · forskolin · heat shock protein · macrophage inflammatory protein 1alpha · macrophage inflammatory protein 1beta · phosphoglycerate mutase · propranolol · prostaglandin E2 · proteome · salbutamol · unclassified drug · zilpaterol · adrenergic system · analytic method · antiinflammatory activity · article · controlled study · data analysis · discriminant analysis · human · human cell · inflammation · liquid chromatography · macrophage · mass spectrometry · multivariate analysis · nucleotide sequence · principal component analysis · priority journal · protein expression · protein secretion · proteomics · statistical analysis · two dimensional difference gel electrophoresis · two dimensional gel electrophoresis · Adrenergic Agonists · Adrenergic beta-Antagonists · Anti-Inflammatory Agents · Biological Markers · Cluster Analysis · Down-Regulation · Electrophoresis, Gel, Two-Dimensional · Enzyme-Linked Immunosorbent Assay · Humans · Immunoassay · Inflammation · Lipopolysaccharides · Macrophage Inflammatory Protein-1 · Macrophages · Mass Spectrometry · Monocytes · Multivariate Analysis · Principal Component Analysis · Propranolol · Proteome · Proteomics · Receptors, Adrenergic, beta-2 · Statistics · Trimethylsilyl Compounds · U937 Cells · Up-Regulation · Escherichia coli

Abstract

Two-dimensional difference gel electrophoresis (DIGE) in combination with univariate (Student's t-test) and multivariate data analysis, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to study the anti-inflammatory effects of the β2-adrenergic receptor (β2-AR) agonist zilpaterol. U937 macrophages were exposed to the endotoxin lipopolysaccharide (LPS) to induce an inflammatory reaction, which was inhibited by the addition of zilpaterol (LZ). This inhibition was counteracted by addition of the β2-AR antagonist propranolol (LZP). The extracellular proteome of the U937 cells induced by the three treatments were examined by DIGE. PCA was used as an explorative tool to investigate the clustering of the proteome dataset. Using this tool, the dataset obtained from cells treated with LPS and LZP were separated from those obtained from LZ treated cells. PLS-DA, a multivariate data analysis tool that also takes correlations between protein spots and class assignment into account, correctly classified the different extracellular proteomes and showed that many proteins were differentially expressed between the proteome of inflamed cells (LPS and LZP) and cells in which the inflammatory response was inhibited (LZ). The Student's t-test revealed 8 potential protein biomarkers, each of which was expressed at a similar level in the LPS and LZP treated cells, but differently expressed in the LZ treated cells. Two of the identified proteins, macrophage inflammatory protein-1beta (MIP-1β) and macrophage inflammatory protein-1 alpha (MIP-1α) are known secreted proteins. The inhibition of MIP-1β by zilpaterol and the involvement of the β2-AR and cAMP were confirmed using a specific immunoassay. © 2005 American Chemical Society.